Analyses that use genome assemblies are critically affected by the contiguity, completeness, and accuracy of those assemblies. In recent years single-molecule sequencing techniques generating long-read information have become available and enabled substantial improvement in contig length and genome completeness, especially for large genomes (>100 Mb), although bioinformatic tools for these applications are still limited.We developed a software tool to close sequence gaps in genome assemblies, TGS-GapCloser, that uses low-depth (∼10×) long single-molecule reads. The algorithm extracts reads that bridge gap regions between 2 contigs within a scaffold, error corrects only the candidate reads, and assigns the best sequence data to each gap. As a demonstration, we used TGS-GapCloser to improve the scaftig NG50 value of 3 human genome assemblies by 24-fold on average with only ∼10× coverage of Oxford Nanopore or Pacific Biosciences reads, covering with sequence data up to 94.8% gaps with 97.7% positive predictive value. These improved assemblies achieve 99.998% (Q46) single-base accuracy with final inserted sequences having 99.97% (Q35) accuracy, despite the high raw error rate of single-molecule reads, enabling high-quality downstream analyses, including up to a 31-fold increase in the scaftig NGA50 and up to 13.1% more complete BUSCO genes. Additionally, we show that even in ultra-large genome assemblies, such as the ginkgo (∼12 Gb), TGS-GapCloser can cover 71.6% of gaps with sequence data.https://github.com/BGI-Qingdao/TGS-GapCloser.TGS-GapCloser can close gaps in large genome assemblies using raw long reads quickly and cost-effectively. The final assemblies generated by TGS-GapCloser have improved contiguity and completeness while maintaining high accuracy. The software is available at  The cost and time necessary to sequence 1 Mb of DNA has been decreasing at a speed beyond Moore's Law over the past decade , 3, syntk-mer extension or local reassembly algorithms but were hindered by large CPU and memory consumption. In addition, these strategies rarely spanned repetitive DNA regions and tended to cause more misassemblies due to the short read lengths of NGS.The first efforts to close gaps in genome assemblies were made using Fosmid and bacterial artificial chromosome libraries combined with Sanger sequencing . But thede novo genome assembly using TGS reads alone is possible, the lower raw read accuracy relative to NGS platforms generally requires sufficient sequencing coverage and high computational costs for error correction of the assembly [Current single-molecule TGS technologies, such as those of Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), have the potential to break through these limitations because their reads can exceed 100 kb and are typically longer than most DNA repeats . Althougassembly . This coassembly .Recently, there have been several hybrid assemblers designed to take advantage of the combination of both TGS and NGS read data. Most construct a final assembly graph by mixing NGS contigs and TGS long reads based on the Overlap-Layout-Consensus or string graph algorithm , or connde novo assembled scaftigs [Three key factors should be considered to develop a TGS gap-closing algorithm. First, use TGS data as little as possible. Although the cost has been decreasing , the gapscaftigs . The misscaftigs , which mde novo assembled human genomes we demonstrate an increase in the scaftig NG50 by 11.0- to 45.0-fold and an increase in the scaftig NGA50 by 6.8- to 30.6-fold. Furthermore, we show that 71.6% of gaps in the ultra-large genome assembly of ginkgo can be closed using just 10.5× coverage of corrected PacBio reads, increasing the scaftig N50 from 57.1 to 364.8 kb. A hybrid strategy of updating a draft de novo genome assembly with TGS-GapCloser is an efficient and accurate strategy for improving the quality of gene annotation and structure variation detection. Ultimately this will help lead to high-quality downstream analyses of ontogeny, phylogeny, and evolution.In this work, we describe a software tool, TGS-GapCloser, that uses low-coverage error-prone long reads to close gaps in large genomes more efficiently and accurately than other current gap-closing tools. Using only 10× coverage of ONT or PacBio long reads , 35 applHomo sapiens  using the MGIEasy stLFR Library Prep Kit on the DNBSEQ-G50 platform (formerly known as BGISEQ-500) , generating a total 660 Gb of read data. Reads mapped to the Chr19 reference were also extracted for comparisons and further analysis. These short reads were assembled using MaSuRCA [RRID:SCR_010691) version 3.3.1 or Mercedes (in-house tool) to obtain short but highly accurate contigs, and the SLR long-range (co-barcode/read cloud) and short-range (paired-end) information provided by the single-tube long fragment reads (stLFR) technique were exploited to do further scaffolding by SLR-superscaffolder [RRID:SCR_016756) was used to obtain draft scaffolds despite being originally designed to assemble 10X Genomics data. To test the potential application of TGS-GapCloser, we used newly generated data from both long-read platforms (ONT and PacBio) to close gaps in human genome assemblies: ONT MinION Rel3 dataset (Rel3) [Three datasets from 2 species containing large genomes were used to examine the gap-closing results by TGS-GapCloser: human, human Chr19, and ginkgo. We sequenced  MaSuRCA  (MaSuRCAaffolder  (versionaffolder  (versiont (Rel3)  and PacBt (Rel3) .Ginkgo biloba (estimated genome size ∼12 Gb) used in this study was obtained from Guan et al. [RRID:SCR_014986) [RRID:SCR_015880) [The genome assembly of a female n et al.  and was _014986)  and upda_014986) . The Pac_015880)  reduced TGS-GapCloser can accept as input any type of TGS long reads or other pre-assembled contigs to fill gaps in a draft assembly in the 4 steps as shown in The input scaffolds were first split into fragments called scaftigs from the observed N positions in the scaffolds, and each pair of neighboring scaftigs based upon their positions in the shared scaffold were defined as a gap to be filled. TGS-GapCloser retains the input scaffold information as the base-level accuracy and the order and orientation of scaftigs, but not the estimated gap size. This is caused by the lack of sufficient resolution in the long-range information provided by SLR, Hi-C, or BioNano to accurately predict the size of gaps below ∼10 kb.RRID:SCR_018550) [We used minimap2   to alignA refers to the alignment length and I refers to the identity ratio for the ith and i+1th scaftigs, respectively; a and b are 2 arbitrary coefficients to distinguish A and I’s weights on the score and have been tuned to 1:6 for the ONT dataset as default. For each gap, a maximum of 10 candidates with the highest QS were chosen for error correction in order to limit the size of data for further analysis. To further reduce the complexity and requirements on computational resources, the overlapped candidates in the same long read were clipped and merged prior to the correction. Either Pilon  [RRID:SCR_017642) [The quantity and quality of candidate reads determine the efficiency and accuracy of gap closure. Thus, we designed a scoring system of candidates for quality control and filtration based on the length and identity ratio  of the alignment between a long-read candidate and flanking scaftig sequence next to the gap. The score QS is given by_017642)  was usedThe corrected candidates were realigned to the gap and scored again, and finally the one with the highest QS was selected to fill the gap. The correction not only increased the single-base accuracy but also helped to find the best final candidate. We hypothesized that the QS of a candidate with higher-quality alignments would be increased due to the more precise mapping to the gap region after error correction, while the candidates with relatively lower-quality alignments tend to fail to be mapped. After final alignment to the gap region, those 2-kb sequences aligning to the scaftigs on either side of the gap were removed and only the bases filling the gap from the highest-scoring candidate were retained.If the highest-scoring candidate resulted in a reduction in bases within the gap, then the gap would collapse to a single scaftig according to the alignment. A portion of scaftigs could have overlaps with other scaftigs because of incorrect paths during the initial assembly graph or over-aggressive scaftig extension. However, a TGS read spanning the gap has the ability to solve the overlap if 2 scaftigs can be mapped to the correct positions. Candidates resulting in a reduction in bases were selected only with more stringent criteria because large indels or homopolymeric repeats in long reads tend to cause incorrect overlaps. Gaps lacking any candidates could not be closed; in some cases this could be due to misassemblies in the draft assembly.TGS-GapCloser is coded in the C++ programing language (requires GCC 4.4+). It uses minimap2 to obtain alignments, and Pilon (requires Java runtime 1.7+) or Racon (requires GCC 4.8+) to correct candidate fragments. The algorithm automatically identifies gaps and tries to find the best matched long-read fragments to close gaps or merge adjacent scaftigs. To accelerate the gap closure without losing efficiency and accuracy, TGS-GapCloser only selects a limited number of fragmented long reads as candidates for subsequent error correction and competition. This also reduces the computational complexity and improves the accuracy through a straightforward but efficient scoring system  and corrThree assemblies and 2 TGS datasets were used to benchmark the utility of TGS-GapCloser in gap closure and scaftig merging in the human genome. Using the same co-barcoded short-read stLFR library, the whole genome was assembled by (i) MaSuRCA-assembled contigs + scaffolds from SLR-superscaffolder, (ii) Mercedes-assembled contigs + scaffolds from SLR-superscaffolder, and (iii) contigs and scaffolds assembled by Supernova using all of the barcoded long-range information. Although MaSuRCA itself can scaffold the contigs, the assembler does not utilize the SLR information and generates relatively short scaffolds. As such, it is necessary to use SLR-superscaffolder to obtain a scaffold NG50 comparable to Supernova.RRID:SCR_001228) [To assess the efficiency of TGS-GapCloser, we used ∼10× coverage of long reads from an ONT Rel3 dataset with a claimed mean read identity of 82.73%  and a Pa_001228)  after gaRRID:SCR_015008) analysis indicated that there are possible improvements for bioinformatics analysis such as gene annotation. The assemblies were compared against the vertebrata_odb9 database. It revealed that 90.5%, 89.7%, and 94.1% of the expected vertebrate genes are complete for Assemblies 1–3, respectively, with ONT Rel3, and 90.4%, 85.3%, and 94.0% for Assemblies 1–3, respectively, with PacBio HiFi. A substantial improvement was observed from the original 86.2%, 76.6%, and 90.7% for Assemblies 1–3, respectively.After gap closure of the assemblies, BUSCO   by the method described by Wenger et al. . The QV olishing , 34.de novo assembly for 30× coverage of long reads requires ∼40,000 CPU hours for ONT and ∼62,000 CPU hours for PacBio [TGS-GapCloser is relatively fast and accurate. For the human genome, it consumed as little as 155 CPU hours in total and 32 GB of peak memory. The algorithm design substantially reduced the time for read mapping and error correction. Gap closure using the NGS-based error correction for the inserted sequences (∼189 hours on average) was much slower than that with the TGS-based correction (∼15 hours on average). As a comparison, the r PacBio . The comk-mer–based extension approaches cannot. In this article, we used a variety of published long-read gap closers, including PBJelly  [RRID:SCR_000646) [We did not compare TGS-GapCloser to NGS gap-closing tools because the use of TGS read information can span the repetitive or other complicated regions in the assembly that _012091) , FGAP [2_012091) , GMclose_000646) , Cobbler_000646) , and LR__000646) , on the The comparison shows that TGS-GapCloser performed best overall among 6 tools with this combination of inputs Table . Its gapIn terms of accuracy, TGS-GapCloser led to the largest increase (>5.2×) in the scaftig NGA50 (16.0 folds to input) with fewer misassemblies. Although FGAP and LR_Gapcloser extended the scaftig NG50 longer than 100 kb, both generated more misassemblies, resulting in a shorter scaftig NGA50. Most gap-closing tools were originally designed for error-corrected long reads or high-quality pre-assembled contigs, and as a result, their performances are mostly unsatisfactory with low-coverage raw ONT reads.In addition, we analyzed the running time and memory consumption for each tool under the same operating conditions. TGS-GapCloser ran approximately 261-, 224-, 1,428-, 2-, and 6-fold faster than PBJelly, FGAP, GMcloser, Cobbler, and LR_Gapcloser, respectively. GMcloser and FGAP (based on BLAST ) were thIt is worth noting the effects of long-read coverage on the gap closure. We randomly extracted 1×, 5×, 10×, 20×, and 29× coverages of mapped ONT Rel3 reads against the Chr19 reference and individually applied them to the same Chr19 MaSuRCA + SLR-superscaffolder assembly by TGS-GapCloser using the same default parameters. As shown in TGS read data have been shown to be useful in the assembly of the human MHC region. This ∼6-Mb region in Chr6 is difficult to assemble with short reads only owing to high repetition and polymorphism . It contThere are potential future improvements to consider for TGS-GapCloser. The selection of inserted sequences largely depends on the performance of the aligner. Although minimap2 performs well in most cases, the alignment results in errors if the pairwise sequences share small overlaps. We believe that this can be solved by using other aligners or additional parameter optimization. In addition, the computational consumption by error correctors or polishers is still significant, even with our efforts to reduce the input data size as much as possible. As error correction tools are updated and ideally become more efficient, TGS-GapCloser performance will benefit from these improvements. In addition, as long-read error rates continue to decrease, as promised by ONT and PacBio, it may be possible to eliminate this extra step of error correction. Finally, we use the input scaffolds including the orientation and order relations of scaftigs to retain the existing assembly information, ignoring possible assembly errors. As a future update we plan to use the information provided by TGS reads to correct scaftig errors within the same scaffold and link different scaffolds if sufficient overlapping is present. Nonetheless, in its current form, TGS-Gapcloser enables the combination of different genetic information with different lengths and resolutions and makes it possible to complete high-quality (ultra) large genome assemblies.RRID:SCR_012091), FGAP (version 1.8.1), GMcloser (version 1.6.2) , Cobbler (version 0.6.1), and LR_Gapcloser (no version information available) . Some were unable to close gaps using the default parameters on low-coverage raw TGS reads. As a result, we needed to tune FGAP to be able to close large gaps . For GMcloser, we used the example parameters for long reads from the manual. In addition, the parameters for Cobbler were tuned according to the authors’ guidance on GitHub. All other tools were run using the default parameters for ONT data.We compared the performance of TGS-GapCloser with that of 5 TGS gap-closing tools, including PBJelly (version PBSuite_15.8.24)  was used to determine length statistics for the assembly such as total length, scaffold NG50, and scaftig NG50, as well as alignment to the reference, including scaffold NGA50, scaftig NGA50, genome fraction, misassemblies, and local misassemblies. To further assess the efficiency and accuracy of TGS-GapCloser, we aligned the reference assembly against the input scaffolds to generate theoretically filled gap sequences using QUAST intermediate files and compared them to the filled sequences by TGS-GapCloser with minimap2 (-x map-ont). Gaps that were capable of being filled by the reference were chosen to evaluate the sensitivity and PPV. Note that gaps smaller than 100 bp were filtered out. The sensitivity is defined as the ratio of the number of TGS-GapCloser–filled gaps that the reference also successfully fills to the total number of gaps that the reference can fill. The PPV is defined as the ratio of the number of TGS-GapCloser–filled gaps that can be uniquely matched to the reference-filled gaps to the total number of filled gaps by both. Note that TGS-GapCloser also completes gaps that the reference cannot fill and as such the accuracy of these cannot be easily determined. The single-base–level accuracy was quantified by mapping the scaftigs in the assembly to the GIAB high-confidence regions in the reference genome GRCh37 to calculate the concordance QV with the method in Wenger et al. [QUAST   into several portions at the position of large gaps (>1 kb) because the aligner tblastn  in BUSCOProject name: TGS-GapCloserhttps://github.com/BGI-Qingdao/TGS-GapCloserProject home page: Operating system(s): LinuxProgramming language: C++, shellOther requirements: Racon, or SAMtools and Pilon are required to be pre-installedLicense: GPLv3RRID:SCR_017633biotools ID: TGS-GapCloserConda access: conda install -c bioconda tgsgapcloserGigaScience GigaDB database [The stLFR sequencing data for the human sample (HG001/NA12878) have been deposited in the CNGB under accession No. CNP0000066. We downloaded the ONT long reads of human from , and Pacdatabase .Supplementary Figure S1: Effects of long-read coverage on gap closure. (A) the number of filled gaps and bases, (B) wall-clock time and peak memory, (C) accuracy in long-read selection, and (D) accuracy at single-base level. All datasets were run with 16 threads.Supplementary Figure S2: Length distribution of gaps in draft scaffolds and that of TGS-GapCloser–filled gap sequences.SupplementaryFigure S3: Read length distribution for the input ONT Rel3 and PacBio HiFi reads.Supplementary Table S1: Summary of the input assemblies in this work.Supplementary Table S2: Summary of the updated assemblies in this work.Supplementary Table S3: The effect of the scoring system on the candidate selection and the gap-closing performance.Supplementary Table S4: The effect of error correction on the candidate selection and the gap-closing performance.SupplementaryTable S5: The effect of long-read coverage on the TGS assemblies and gap-closing results.SupplementaryTable S6: Genomics dataset source.Supplementary Table S7: Control parameters used for different software tools.giaa094_GIGA-D-20-00014_Original_SubmissionClick here for additional data file.giaa094_GIGA-D-20-00014_Revision_1Click here for additional data file.giaa094_GIGA-D-20-00014_Revision_2Click here for additional data file.giaa094_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giaa094_Response_to_Reviewer_Comments_Revision_1Click here for additional data file.giaa094_Reviewer_1_Report_Original_SubmissionChong Chu -- 3/19/2020 ReviewedClick here for additional data file.giaa094_Reviewer_1_Report_Revision_1Chong Chu -- 6/7/2020 ReviewedClick here for additional data file.giaa094_Reviewer_2_Report_Original_SubmissionJustin Chu -- 3/20/2020 ReviewedClick here for additional data file.giaa094_Reviewer_2_Report_Revision_1Justin Chu -- 6/10/2020 ReviewedClick here for additional data file.giaa094_Supplemental_FileClick here for additional data file.BLAST: Basic Local Alignment Search Tool; bp: base pairs; BUSCO: Benchmarking Universal Single-Copy Orthologs; Chr19: Chromosome 19; Chr6: Chromosome 6; CNGB: China National GeneBank; CPU: central processing unit; Gb: gigabase pairs; GIAB: Genome in a Bottle; kb: kilobase pairs; Mb: megabase pairs; MHC: major histocompatibility complex; NGS: next-generation sequencing; ONT: Oxford Nanopore Technologies; PacBio: Pacific Biosciences; PPV: positive predictive value; QS: quality score; QV: quality value; SIMD: single-instruction-multiple-data; SLR: synthetic long reads; stLFR: single-tube long fragment reads; TGS: third-generation sequencing.All authors are employees of BGI or its subsidiaries. The authors declare that they have no other competing interests.This research was supported by the Shenzhen Municipal Government of China Peacock Plan (No. KQTD2015033017150531), the National Key Research and Development Program of China (Grant No. 2018YFD0900301-05), and the Qingdao Applied Basic Research Projects (Grant No. 19-6-2-33-cg).M.X., L.G., and L.D. performed software design and implementation. M.X., L.G., S.G., O.W., and R.Z. contributed to data modeling, data curation, and assembler benchmarking. M.X. wrote the draft manuscript, and L.G., B.A.P., G.F., L.D., Y.Z., X.L., and X.X. contributed to manuscript editing. L.D. and Y.Z. supervised the project. M.X., G.F., and Y.Z. secured funding. All authors read and approved the final manuscript.


Different from adult clinical stage I (CS1) testicular cancer, surveillance has been recommended for CS1 pediatric testicular cancer. However, among high-risk children, more than 50% suffer a relapse and progression during surveillance, and adjuvant chemotherapy needs to be administered. Risk-adapted treatment might reduce chemotherapy exposure among these children.A decision model was designed and calculated using TreeAge Pro 2011 software. Clinical utilities such as the relapse rates of different groups during surveillance or after chemotherapy were collected from the literature. A survey of urologists was conducted to evaluate the toxicity of first-line and second-line chemotherapy. Using the decision analysis model, chemotherapy exposure of the risk-adapted treatment and surveillance strategies were compared based on this series of clinical utilities. One-way and two-way tests were applied to check the feasibility.In the base case decision analysis of CS1 pediatric testicular cancer, risk-adapted treatment resulted in a lower exposure to chemotherapy than surveillance (average: 0.7965 cycles verse 1.3419 cycles). The sensitivity analysis demonstrated that when the relapse rate after primary chemotherapy was ≤ 0.10 and the relapse rate of the high-risk group was ≥ 0.40, risk-adapted treatment would result in a lower exposure to chemotherapy, without any association with the proportion of low-risk patients, the relapse rate of the low-risk group, the relapse rate after salvage chemotherapy or the toxicity utility of second-line chemotherapy compared to first-line chemotherapy.Based on the decision analysis, risk-adapted treatment might decrease chemotherapy exposure for these high-risk patients, and an evaluation after orchiectomy was critical to this process. Additional clinical studies are needed to validate this statement. Despite the low incidence of pediatric testicular tumors, yolk sac tumors are the most common malignant type in children, which are very different from their adult counterparts –6. ApproIn recent studies, approximately 20% of children with CS1 testicular germ cell tumors (GCT) have suffered a relapse within 4 years after RIO, and they underwent 3–4 cycles of salvage chemotherapy . AdvanceIn this study, using a decision analysis model, we evaluated the chemotherapy burden of CS1 pediatric testicular cancer between risk-adapted treatment and surveillance.http://www.treeage.com), and the decision trees of the surveillance and risk-adapted treatment and flowchart of the analysis are listed in Figs. The decision model was designed and calculated using TreeAge Pro 2011 Software  chemotherapy. If a complete response (CR) was not achieved after 3 cycles of PEB, second-line chemotherapy with 3 cycles of VIP  was performed. For the risk-adapted group, the high-risk group received primary chemotherapy with 1 cycle of PEB, and the low-risk group underwent surveillance. Then, salvage chemotherapy with 3 cycles of PEB was performed when a relapse was detected. If a CR was not achieved after 3 cycles of PEB, second-line chemotherapy with VIP was performed.According to recent studies, the overall survival of CS1 pediatric testicular cancer was nearly 100% with systemic chemotherapy, and the progression rates after primary and salvage chemotherapy were both approximately 5% 2.3–6.8%)  and 0.15 0.10–0.20), respectively; the proportion of low-risk patients was 0–1, the progression rates after primary and salvage chemotherapy were both 0.05 (0.01–0.10), and second-line chemotherapy was the last treatment with a 100% success rate  and 0.635 , respectively. Therefore, we defined the toxicity of second-line chemotherapy as approximately 0.814/0.635 = 1.3 times that of salvage chemotherapy. The range was defined as 1.0–2.0 in the decision analysis.In all, 24 urologists and oncologists took part in the interview to evaluate the toxicity of chemotherapy for pediatric testicular cancer. As shown in Table Our analysis demonstrated that risk-adapted treatment resulted in a lower exposure to chemotherapy than surveillance (average: 0.7965 cycles verse 1.3419 cycles). A 1-way sensitivity analysis demonstrated that the differences in chemotherapy exposure between the two treatments were associated with the proportion of low-risk patients (pLowRisk): when pLowRisk = 0, all of the patients were in the high-risk group, and the two treatments had significantly different exposures to chemotherapy; when pLowRisk = 1, all patients were in the low-risk group, and the two groups had the same exposure to chemotherapy  and the relapse rate after primary chemotherapy (pRelapsePostPrimChemo) were associated with chemotherapy exposure. When pRelapseHighrisk was ≥ 0.40 or pRelapsePostPrimChemo was ≤ 0.25, risk-adapted treatment resulted in lower chemotherapy exposure, and these two utilities are reasonable in clinical practice Fig. . Within In recent studies, the rate of relapse was approximately 20% for CS1 pediatric testicular cancer, and most cases occurred in the first 2 years . In someActually, based on the contemporary scenario, this study revealed that risk-adapted treatment was associated with significantly less chemotherapy exposure. pRelapsePostPrimChemo and pRelapseHighrisk were significant factors that decreased exposure to chemotherapy, which implied that the effectiveness of primary chemotherapy and the identification of high-risk patients were critical to individualized management. For primary chemotherapy, the outcome is favorable and a lower-toxicity regimen might be available . In a reOur study had some limitations worth noting. To simplify the analysis of chemotherapy toxicity, we calculated cycles of chemotherapy instead of the detailed side effects, such as cardiovascular disease, neurotoxicity, ototoxicity, chronic kidney disease, and infertility. The proportions were defined according to recent studies, but since these cases are rare, bias was present, and some of them were included in studies about their adult counterparts. Due to the shortage of life-long follow-up of this curable disease, quality-adjusted life-years and cost-effectiveness analyses were not performed in this study. Despite these limitations, we believe this model could imply some advantages of risk-adapted management in CS1 pediatric testicular cancer. This is the first report regarding the chemotherapy burden of CS1 pediatric testicular cancer.Our decision model of management for clinical stage 1 pediatric testicular cancer demonstrated that risk-adapted treatment was associated with a lower exposure to chemotherapy. Additional clinical studies are needed to validate this statement.Additional file 1: Figure 1. Way sensitivity analysis. A: In any value of pRelapseLowrisk (relapse rate of low-risk group), when pRelapseHighrisk (relapse rate of high-risk group) > 0.4, risk-adapted treatment was associated with lower exposure of chemotherapy; B: In any value of pRelapsePostSalvChemo , when pRelapseHighrisk (relapse rate of high-risk group) > 0.4, risk-adapted treatment was associated with lower exposure of chemotherapy; C: In any value of tSecondChemo , when pRelapseHighrisk (relapse rate of high-risk group) > 0.4, risk-adapted treatment was associated with lower exposure of chemotherapy. Red: risk-adapted treatment, Blue: surveillance.


The 635-nm laser parameters; dose: 10 J per point (20 J/cm2), time: 100 s per point, two points , the total energy per session 20 J. Laser application protocol: immediately and 3, 6, 9, 12, 15, and 30 days after surgery. The total energy after all therapeutic sessions was 140 J. The implants’ stability was measured employing a Periotest device  immediately and 3, 6, 9, 12, 15, 30, and 60 days after the insertion of the mini-implants. Results: We found significantly higher secondary stability, lower mean PTV (6.18 ± 5.30) and (1.51 ± 2.25), for self-drilling mini-implants  in contrast to the control, G2 group (9.17 ± 8.25) and (5.00 ± 3.24), after 30 (p = 0.0003) and 60 days (p = 0.0000). Moreover, the analysis of the mini-implants stability after 635-nm diode laser application revealed significant higher stability in comparison with none irradiated implants (G2 group) after 3 days. (p = 0.0000) There was no significant difference in pain level measured on the NRS-11 scale on both sides of the maxilla. (p = 0.3665) An important finding was that all inserted mini-implants survived during a two-month observation period. Conclusions: 635-nm diode laser at laser irradiation increases the secondary stability of orthodontic mini-implants.Background: The study aimed to clinically estimate an influence of a 635 nm diode laser on the stability of orthodontic mini-implants, to assess mini-implants loss, and to evaluate a pain level after the treatment. Materials and Methods: The randomized clinical split-mouth trial included 20 subjects , 40 implants  with a diameter 1.4 mm and length of 10 mm. Mini-implants were placed in the area of the attached gingiva between the second premolar and first molar teeth, 2 mm below the mucogingival junction of both sides of the maxilla. Each implant on the right side (G1,  Orthodontic microimplants are increasingly used in orthodontic treatment . Thanks The volume and quality of the bone that affect the osseointegration process are the most critical factors responsible for the long-term clinical success in implant dentistry . MoreoveIn implant dentistry and mini-implant assisted orthodontics, a sufficient process of measuring of implant’s stability, and bone density is essential . BecauseA method that can be applied to ameliorate the process of bone healing and to increase the stability of implants is a low-level laser therapy (LLLT) . The LLLTo date, there has been minimal research investigating the effect of PBM on orthodontic mini-implants’ stability ,21. HoweClinicalTrials.gov (identifier: NCT04175405).The trial was designed as a randomized and controlled test. The approval of the Local Ethics Committee of Wrocław Medical University, Faculty of Dentistry was obtained (permission number: KB-278/2018) and an informed consent in accordance with the Helsinki Declaration was obtained from all participating subjects. The clinical trial was registered with d = 0.58 [The study involved an insertion of 40 orthodontic mini-implants in total, in the posterior region of a maxilla in 20 patients  . Sample d = 0.58 . All of Patients qualified for the study were treated with a straight wire technique using fixed appliances with MBT .022″ prescription .A total number of 40 orthodontic mini-implants , made of titanium alloy (grade 5), 10 mm long with a diameter of 1.4 mm, were placed in the area of the attached gingiva between second premolar and first molar teeth 2 mm below mucogingival junction of both sides of maxilla. The soft tissue was removed using a ceramic bur, and each implant was placed immediately with a hand driver without bone decortication.  After th2, average power density: 199.04 mW/cm2, continuous mode, dose: 10 J per point (20 J/cm2), time: 100 s per point, 2 points , total energy per session 20 J. The diode laser was used in contact mode with peri-implant soft tissue on the right side of the maxilla (group G1) according to the following irradiation protocol: immediately and 3, 6, 9, 12, 15, 30 days after surgery. The total energy after all therapeutic sessions was 140 J.  at 635-nm wavelength at the following set parameters; output power: 100 mW, handpiece diameter: 8 mm, spot area: 0.5024 cms 140 J.  The subjThe mini-implants’ stability was estimated employing a Periotest device . The Periotest measurement system includes the sound formed from contact between an object and a metallic tapping bar in a handpiece, which is electromagnetically driven and electronically verified. The Periotest response detection is analyzed through a fast Fourier transform (FFT) algorithm. Simply put, the Periotest answer to tapping is estimated by an accelerometer and then analyzed. The signal generated by tapping is then transformed to a value called the Periotest value (PTV), which depends on the damping characteristics of the peri-implant tissue . The PerImmediately after the mini-implants’ placement, each patient received a questionnaire for individual pain assessment . The maximum pain level was measured on both sides of the maxilla during the first day after the treatment. The NRS-11 scale consists of a conscious, subjective assessment of the pain experienced; therefore, it is used in the case of patients over ten years old. A rating of 0 signifies no pain, 1–3 represents mild pain, 4–6 moderate pain, and 7–10 severe pain .t-test using the Statistica 12 software  at a significance level of p = 0.05.To assess whether the data were normally distributed, the Kolmogorov–Smirnov test was performed at the 95% level. Differences in mini-implants’ stability and pain value were compared with the dependent sample Student’s p = 0.0003) and 60 days. (p = 0.0000)  and (1.51 ± 2.25), for self-drilling mini-implants  in contrast to the control, G2 group (9.17 ± 8.25) and (5.00 ± 3.24), after 30 ( 0.0000) .p = 0.0000)  in comparison with no irradiated mini-implants (PTV–1.05 ± 1.13) after three days ( 0.0000) .p = 0.3665)  .An important finding was that all inserted mini-implants survived during the two months observation period.2 accounted to significantly greater secondary stability  in contrast to non-irradiated side. Furthermore, a similar score of pain level was recorded on both sides of the maxilla, which agrees with findings of other randomized clinical split-mouth study concerning the effect of the application of low-level laser with 4-Joule or 16-Joule energy on pain reduction following elastomeric separators placement [The PBM is a minimal-invasive treatment allowing to stimulate cellular processes such as ATP, DNA, and RNA synthesis ,22. Manylacement .2 is optimal to obtain an optimal biological answer [2) allowed increasing the secondary mini-implant stability.Photobiomodulation therapy in a wavelength spectrum of 600–1100 nm  appears in a more extensive penetration depth what causes a more significant cell-light answer . LLLT efl answer . In our The interesting findings of our present study was a slower trend of reduction in implant stability for irradiated mini-implants during the first two weeks as compared to the control group. This phenomenon could be explained by the effect of the red wavelength on the inflammation phase of the bone after the injury evoked by placing the implants . In the 2, 135 sec·2 points; the mesial and distal sides of the mini-implant, 54 J per session) group in contact to non-irradiated mini-implants in a rat model. It should be highlighted that in the study of Omsa et al. the authors used higher energy dose per session in contrast to our present study and out of the optical window range (1–10 J/cm2) described by Arndt-Schulz curve. The positive results of PBM in mini-implants stability were confirmed also by the study published by Pinto et al. [To date, no randomized split-mouth controlled trial before in humans has evaluated the role of low-level laser energy on mini-implants stability in orthodontics. However a few studies concerning the use of LLLT on orthodontic mini-implants stability were commenced. ,21 In thThe second aim of our study was to determine the effect of the LLLT on pain level in the same day when placed orthodontic mini-implants. In our study there was no significant difference in pain level measured on the NRS-11 scale on both sides of the maxilla. In the scientific literature, there is no other evidence proving our findings. However, in the split-mouth clinical study presented by AlSayed et al., the authors obtained no difference in relieving elastomeric separators induced orthodontic pain after LLLT, applied at two different laser energy values (4 and 16 J). However, there is some bias in pain analysis in this study because the pain feeling by the patients could alsom be caused by the fixed orthodontic appliance . There iFurthermore, the mini-implants’ failure rate (mini-implant loss) for a 60-day observation period was evaluated. In our present study, all thirty implants survived and serve as an anchorage system in the orthodontic treatment. Our previous in vitro study  in mandiNecessary attention should be paid to preventing the thermal injury of the periodontal tissues when irradiating tissues with lasers ,31,32,33Additional studies using PBM and mini-implants applied in orthodontics are required to determine long-term clinical benefit in the described method. To evaluate the influence of the LLLT (red and infrared wavelengths) on peri-implant bone tissue, additional randomized-controlled trials in the long-term and on larger study groups are recommended.Irradiation of peri-implant soft tissue using a 635-nm diode laser enhances secondary mini-implant stability after three days, one month, and two months. The diode laser application has no significant effect on pain level after orthodontic appliance placement measured in the NRS-11.


Patients and Methods. We enrolled 18 EPS patients and 90 gender-matched PD patients without EPS in this cross-sectional case-controlled study. The effluent MMP-2 levels and possible risk factors for EPS were analyzed using multivariable logistic regression, and a nomogram was developed. The nomogram was validated using 200 bootstrap resamples to reduce overfit bias.  Encapsulating peritoneal sclerosis (EPS) is a rare but serious complication of peritoneal dialysis (PD). So far, there is no biomarker-based prediction tool available for EPS. Matrix metalloproteinase-2 (MMP-2) is a protein involved in the breakdown of the extracellular matrix, and the effluent MMP-2 can be a potential biomarker of EPS. This study is aimed at developing a nomogram for EPS based on effluent MMP-2 levels. p < 0.001, Manny-Whitney U test). Effluent MMP-2 levels and PD duration were independently associated with EPS risks (p < 0.001 and p = 0.001) in multivariate logistic regression. A nomogram based on MMP-2 levels and PD duration was proposed. The AUC of MMP-2 was 0.824, and the AUC of the nomogram was 0.907 (p = 0.05).  The effluent MMP-2 levels in EPS patients were significantly higher than those in normal PD patients ( A nomogram based on effluent MMP-2 levels and PD duration may predict EPS with high accuracy. Encapsulating peritoneal sclerosis (EPS) is a rare but serious complication of peritoneal dialysis (PD) , 2. The The recruitment and follow-up protocols complied with the Declaration of Helsinki and were approved by the institutional review board of the China Medical University Hospital (CMUH104-REC2-045), and written informed consent was obtained from each individual. We enrolled 18 EPS patients from three tertiary hospitals in Taiwan from Jan. 2012 to Dec. 2017. The diagnosis criteria of EPS included bowel obstruction, ascites, and blood-stained effluent in combination with a loss of net ultrafiltration [μg/mg) were standardized for per mg/dl of total protein in the effluent. None of the dialysate MMP-2 levels was below the detection limit. There were no missing data.We collected peritoneal effluents from EPS patients at the diagnosis of EPS and at the time of the peritoneal equilibrium test (PET) in control patients. The samples were collected after a 4 h dwell time and were frozen at a temperature of −80°C within 24 h. The peritoneal effluents were sent to a central laboratory for MMP-2 measurements using the Human MMP-2 ELISA kit (Invitrogen: #KHC3081) according to the manufacturer's protocol. Effluent MMP-2 levels , medians , or frequencies (percentages) where appropriate. All continuous variables were tested for normality using the skewness and kurtosis test. Data were analyzed using the y et al. . The numy et al. . Possiblp = 0.032) than the control patients . The serum levels of albumin, calcium, and phosphorus were not different between the control patients and the EPS patients. The serum creatinine levels (8.74 ± 2.61 mg/dl) of EPS patients were lower than that  of the control patients. The percentage of patients with high transport in PET was higher in EPS patients than in control patients.EPS patients were younger  of the control patients. The effluent MMP-2 levels significantly correlated with EPS based upon univariate and multivariate logistic regressions  and were significantly higher than those  based upon the multivariable logistic regression. Patients' ages, serum phosphorus, albumin, calcium, creatinine, and PET were not associated with EPS in multivariable logistic regression and were not included in the nomogram. A nomogram including effluent MMP-2 levels and duration of PD was proposed based on the results of the multivariate logistic regression (p = 0.05). Ultimately, the AUC of the nomogram was 0.907 for the diagnosis of EPS.The ORs were 156 (95% CI: 18.1–1350) per log unit of MMP-2 in the univariate logistic regression and 74.7 (95% CI: 7.36–758) per log unit of MMP-2 in the multivariate logistic regression. The log-transformed MMP-2 levels were used in the logistic regressions because the distribution of MMP-2 was skewed. Longer duration of PD was associated with higher risks of developing EPS with an OR of 1.28 (95% CI: 1.03–1.60) for each additional year  and is on PD treatment for 12 years (which corresponds to 20 points on the “points” line). The total scores are 80 points that equal to 97.5% risk of EPS (0.975 on the risk of EPS axis). The physicians may decide on further investigations such as abdominal CTs, laparoscopy, holding PD treatment, and delayed removal of the PD catheter. Delayed removal of the PD catheter may facilitate the diagnosis of EPS because 45% of the patients developed EPS after stopping PD treatment [The standardization of MMP-2 levels using total effluent protein levels is critical because the overall solute transport can change in EPS patients , 13. MMPThe peritoneal function such as water transport can be restricted in EPS , 20, butThere are some limitations to this study. First, some factors that may be associated with the development of EPS were not examined in this study, such as the use of PD solutions containing high concentrations of glucose. Second, markers of chronic inflammation, such as C-reactive protein or IL-6 , were noHigher effluent MMP-2 levels are associated with an increased risk of EPS in long-term PD patients. A simple nomogram that consists of effluent MMP-2 levels and PD duration may be associated with EPS with high accuracy.


Xx1−YZ, with different degrees of short- and long-range vacancy order can be explained using a simple model. In this model, the vacancies avoid being nearest or next-nearest neighbors. The model reproduces previously observed diffuse electron-diffraction data. The findings suggest a method for further improving thermoelectric efficiency of these materials.Defective half-Heuslers,  Xx1−YZ with large amounts of intrinsic vacancies, such as Nbx1−CoSb, Tix1−NiSb and Vx1−CoSb, are a group of promising thermoelectric materials. Even with high vacancy concentrations they maintain the average half-Heusler crystal structure. These systems show high electrical conductivity but low thermal conductivity arising from an ordered YZ substructure, which conducts electrons, while the large amounts of vacancies in the X substructure effectively scatters phonons. Using electron scattering, it was recently observed that, in addition to Bragg diffraction from the average cubic half-Heusler structure, some of these samples show broad diffuse scattering indicating short-range vacancy order, while other samples show sharp additional peaks indicating long-range vacancy ordering. Here it is shown that both the short- and long-range ordering can be explained using the same simple model, which assumes that vacancies in the X substructure avoid each other. The samples showing long-range vacancy order are in agreement with the predicted ground state of the model, while short-range order samples are quenched high-temperature states of the system. A previous study showed that changes in sample stoichiometry affect whether the short- or long-range vacancy structure is obtained, but the present model suggests that thermal treatment of samples should allow controlling the degree of vacancy order, and thereby the thermal conductivity, without changes in composition. This is important as the composition also dictates the amount of electrical carriers. Independent control of electrical carrier concentration and degree of vacancy order should allow further improvements in the thermoelectric properties of these systems.Defective half-Heusler systems  At room temperature, the Cu atoms are ordered in two dimensions but disordered along the third dimension, and above ∼400 K the Cu substructure becomes almost liquid like  calculations . In the HHL plane (spanned by HH0 and 00L) continuous waves of diffuse scattering were observed between rows of Bragg peaks along the 00L direction, as sketched in Fig. 1b). In the Nbx1−CoSb system, Xia et al. also observed several ordered structures depending on the nominal stoichiometry of the samples .For the samples with nominal stoichiometry Nbet al. (2019)X substructure of the structure.As very similar types of short-range order diffuse scattering have been observed in several different half-Heusler systems it seems to be a common feature of these systems. Here we give a simple model for both the diffuse scattering as well as the ordered structures observed by electron diffraction by Xia 2.Xx1−YZ half-Heusler systems is shown in Fig. 2a). The half-Heusler structure has X on a face-centered cubic (f.c.c.) lattice with Z in the octahedral positions and Y in half of the tetrahedral positions. X and Y together form the sphalerite structure while X and Z together form the rock-salt structure. As these systems have a deficiency of X, there will be vacancies in the structure. It was found that there are no apparent anti-site defects in these systems .The average structure of the defective a). Here the gray circles show the sites with whole integer z coordinates and the gray triangles show the sites with half-integer z coordinates. In this view, the unit-cell axes are along the diagonal, as marked by the red square. This representation is convenient for representing the ordered vacancy structures as shown below.The question of the atomic short- and long-range order can therefore be simplified to how the vacancies are distributed on the f.c.c. lattice. To illustrate different vacancy distributions we will use a different representation of this structure, where the f.c.c. substructure is viewed from above, as shown in Fig. 3X substructure it is not possible to arrange the vacancies such that there are no nearest neighbors. When x = 1/4 it is possible to make an ordered arrangement with no nearest neighbors, but with each vacancy having four out of six next-nearest neighbors also vacant. This arrangement is illustrated in Fig. 3b). In order to both completely avoid nearest and next-nearest neighbor vacancies, 1/6 sites need to be vacant. Several possible arrangements will satisfy these conditions, and one such arrangement is shown in Fig. 3d). More are shown in the Supporting information. This means that for x < 1/4 nearest neighbors can be eliminated and for x < 1/6 also all next-nearest neighbors can be eliminated. In cases where 1/4 > x > 1/6 there has to be some next-nearest neighbor vacancies if nearest-neighbor vacancies are still to be avoided.We make the simple assumption that vacancies avoid each other, and only take into account nearest and next-nearest neighbor interactions. When there are more than 1/4 vacancies in the x = 1/5 suggested by Zeier et al. (2017c). In this structure, each vacancy has no nearest neighbors but two next-nearest neighbors. However, if instead of adapting the ordered x = 1/5 structure the vacancy substructure creates domains of x = 1/6 and x = 1/4 type structures there will be a lower average number of next-nearest neighbors. This will allow 3/5 of the vacancies to go into the x = 1/6 structure, leaving 2/5 of the vacancies in the x = 1/4 structure. This will give an average number of next-nearest neighbors per vacancy of 1.6, somewhat lower than in the structure suggested by Zeier et al.  sites of vacancy i. Sij is 1 if site j has a vacancy and 0 if it is occupied. Similarly, the sum over j′ is over the six next-nearest neighbors (NNN) of vacancy i, where Sij′ is 1 if site j′ is vacant and 0 if it is occupied. In this formalism, an energy of J1 is assigned to a nearest-neighbor vacancy pair and J2 for a next-nearest neighbor vacancy pair.For 3.Python. A box of 24 × 24 × 48 sites with the appropriate number of vacancies was started in either a random configuration or from one of the ordered states. In cases where the ordered starting state was used for a different x than that of the ordered state, either additional vacancies were added randomly or random vacancies were removed in order to have the correct number of total vacancies. Vacancies were allowed to move to nearest-neighbor positions following the Metropolis algorithm  and 4(b) for the 0KL and HK0 planes, which are symmetry equivalent in the average cubic structure. The 0KL plane shows additional peaks corresponding to those Xia et al. observed for their Nb0.81CoSb sample  is obtained. Now the 8-point rings have emerged, which are very similar those observed in several samples by Xia et al.  and HHL planes (lower row) which have been symmetry averaged to the Laue symmetry of the average cubic structure. The scattering from the ordered x = 1/4 structure presented in Fig. 3b) is shown in the first column of Fig. 5HK0 plane are the rows of additional peaks along odd-integer H and K. The scattering of the ordered x = 1/6 structure presented in Fig. 3d) is shown in the third column of Fig. 5HK0 plane has the 8-point ring without a peak at the center. In the region 1/4 > x > 1/6, the structure obtained from Monte Carlo simulation has scattering which has both features from the x = 1/4 and x = 1/6 structures with additional diffuse scattering. This is shown in the second column of Fig. 5x = 1/5. The 8-point rings here also have peaks at their center and there are additional peaks between the main reflections. In this model there are inclusions of the x = 1/4 type regions in the x = 1/6 type structure. The scattering of this model matches that reported by Xia et al. for samples with nominal stoichiometry Nb0.82CoSb and Nb0.84CoSb [measured stoichiometry Nb0.81CoSb and Nb0.82CoSb . The structure suggested by Zeier et al. for x = 1/5 based on DFT calculations  and 1b). The same high-temperature short-range order scattering is also obtained when the stoichiometry is different, as shown in the Supporting information. The x = 1/10 ground-state scattering, shown in Fig. 5Fig. S1 in the Supporting information). This is because of the inherent disorder in all x < 1/6 systems as there are many states all obeying no nearest and next-nearest neighbor vacancies.As the temperature of the simulation is increased, the long-range order disappears, leaving the short-range order. First, the peaks corresponding to the inclusions of the ordered  al. 2019, as sketJ2/J1 = 0.48 for the relative energy penalty for a next-nearest neighbor compared with a nearest-neighbor vacancy pair, as this reproduces the observed scattering. The calculated diffuse scattering for other ratios is shown in Figs. S4 and S5.All the simulations shown here used the ratio 5.et al. on different samples can all be explained through the simple model presented above for vacancy distribution in Xx1−YZ systems. The short-range order scattering from Ti0.9NiSb, and V0.9CoSb matched the simulations with x = 1/10, as shown in Fig. 50.81CoSb [measured stoichiometry Nb0.80CoSb  and 1b). If the octahedral cluster model is also used to explain the scattering observed for the short-range order in the half-Heusler systems, the octahedra in the f.c.c. substructure should be as close as possible to the average stoichiometry. An example of one such octahedra is the six side-centered atoms of the f.c.c. unit cell. However, every site of the f.c.c. substructure is contained in six octahedra. Those six octahedra are made from all nearest and next-nearest neighbors to the site. For x ≤ 1/6, this model would imply that all octahedra have at most one vacancy. This is equivalent to all vacancies having no nearest or next-nearest neighbors, as is the case for the ground states found in this study. For 1/4 > x > 1/6, each octahedra would sometimes contain two vacancies in the cluster model. Although this is also true for the ground states found here, the two are not equivalent, as the cluster model will allow some nearest neighbors, while the found ground states do not have nearest-neighbor vacancies, only some next-nearest neighbors in this range. However, the high-temperature states found here for 1/4 > x > 1/6, for which the scattering is shown in Fig. 6The diffuse scattering patterns of the short-range order states, as seen in Fig. 6et al. observed that the alloy Ni4Mo changes structure when annealed (De Ridder et al., 1976x = 1/5 structure suggested by Zeier et al. (20173Mo a diffraction pattern with the 8-point ring was obtained, similar to the ones shown here. These ordered systems can also be explained in terms of the octahedral cluster model (De Ridder et al., 1976Some previously reported systems also show similar types of ordering as the half-Heusler samples. De Ridder  al. 2017 shown inet al. (2010et al. (2016The short-range order observed in these half-Heusler systems therefore belongs to a more general type of short-range order found in many systems with substitutional or vacancy disorder in f.c.c. substructures. Although these systems are chemically very different, the rule that local stoichiometry should be conserved leads to the same topology of the diffuse scattering. Indeed there are many such families of equivalent short-range order found in chemically very diverse systems (Keen & Goodwin, 2015 al. 2010. The dif al. 2016. Recentl al. 2016. This me al. 2016.6.Xx1−YZ with large amounts of vacancies have previously been found to show interesting scattering patterns, indicating short-range order in some samples and long-range order in others. Here, we have shown that all observed scattering patterns can be explained in terms of a simple model. The model assumes that vacancies tend to avoid being close to each other. The vacancies are distributed on a face-centered cubic substructure of the half-Heusler structure. By avoiding nearest-neighbor vacancy pairs and minimizing next-nearest neighbor vacancy pairs, the observed ordered phases can be explained. Furthermore, by simulating the effect of temperature on the system, the observed short-range order phases can be explained as quenched non-ground-state phases. It is also found that some of the reported measurements come from a single orientation of the ground-state phase, while other reported measurements originate from several ground-state domains with different orientations.Defective half-Heusler systems As the observed short-range order state can be explained through this model as a quenched high-temperature state, while the observed ordered phase is the ground state of the same system, it suggests that the occurrence of the two phases can be controlled through thermal treatment of samples. Such control of the degree of disorder/order in the system should in turn allow tunable thermoelectric properties, as the thermal conductivity is expected to depend on the degree of long-range order. It has previously been observed that changes in sample composition can affect whether short- or long-range vacancy ordering is obtained. However, the composition also dictates the number of electrical carriers. This study therefore suggests that the degree of vacancy order, and thereby the thermal conductivity, can be controlled independently of carrier concentration using thermal treatment, allowing further optimization of thermoelectric properties.10.1107/S2052252520005977/fc5045sup1.pdfSupporting information. DOI:


PROM is one of the common complications of perinatal period, which seriously threatens the mother and newborn. The purpose of this study was to identify the role of NLRC4 inflammasomes in this process and their underlying mechanisms. We performed high-throughput RNA sequencing of fetal membrane tissue from 3 normal pregnant women and 3 term-premature rupture of fetal membrane (TPROM) patients who met the inclusion criteria, and found that NLRC4 was significantly up-regulated in TPROM patients. An observational study of TPROM patients  and normal pregnant women  was performed at the Xuzhou Maternal and Child Health Hospital affiliated to Xuzhou Medical University from May 2018 to May 2019. The expression of genes involved in inflammasome complex including NLRC1, NLRC3, AIM2, NLRC4, ASC, caspase-1, IL-6, IL-18 and IL-1βwas determined via real-time PCR, immunohistochemistry and immunofluorescence. Measurement of NLRC4 level in serum was conducted by ELISA assay. The results showed that the NLRC4, ASC, caspase-1, IL-1β and IL-18 levels in fetal membrane, placental tissues and maternal serum were markedly higher in the PROM group than that in the control group. In conclusion, NLRC4 is a markedly up-regulated gene in TPROM fetal membrane tissue, suggesting that NLRC4 is involved in the occurrence and development of TPROM; NLRC4 levels in maternal blood serum are closely related to TPROM and have the potential to assist doctors in predicting and diagnosing PROM. Premature rupture of membranes (PROM) refers to the rupture of the membranes before delivery, which is characterized by painless fluid leaking from the vagina. It is one of the common complications of perinatal period, which seriously threatens the mother and newborn –3. PROM A growing body of evidence supports the concept that physiological and pathological inflammation during pregnancy is linked to the innate immunity. Innate immune receptors, such as C-type lectin receptors (CLR), nucleotide oligomerization domain (NOD)-like receptors (NLR), and absent in melanoma 2 (AIM2)-like receptors (ALR), serve as the first line of defense against infectious microorganisms, constantly monitoring the extracellular milieu and subcellular compartments –7.Bryant AH et al [NLRs are activated by pathogen-associated molecular patterns or by danger-associated signals . The NLRAH et al . confirmThe purpose of this study was to identify genes that are differentially expressed in patients with TPROM by a genome-wide approach, and to further explore the role of NLRC4 inflammasomes in this process and their underlying mechanisms.This study included 60 pregnant women who delivered at the Xuzhou Maternal and Child Health Hospital affiliated to Xuzhou Medical University from May 2018 to May 2019. Group1 (PROM group) consisted of 30 women with TPROM. Group2 (control group) consisted of 30 women with full-term gestational age≥37 weeks and <42 weeks. All these women had normal outcomes .All selected women met the following criteria: (a) singleton pregnancy; (b) full-term pregnant women planning to give birth in our hospital. Women with the following conditions were excluded: (a) Women who did not meet the inclusion criteria; (b) patients with severe pregnancy complications; (c) women with neonates having congenital or chromosomal abnormalities. All women provided informed consent before collection of placental and membrane tissue, and the study was approved by the Clinical Research Ethics Committee of affiliated Hospital of Xuzhou Medical University. We declared that all methods in this study were carried out in accordance with relevant guidelines and regulations.The fetal membrane tissues from 3 normal pregnant women and 3 TPROM patients who met the criteria for inclusion and not combined with histological chorionitis (HCA) were stored in RNA later solution for future study. RNA high throughput sequencing was performed by Cloud-Seq Biotech . Briefly, total RNA was used for removing the rRNAs with NEBNext rRNA Depletion Kit  following the manufacturer's instructions. RNA libraries were constructed by using NEBNext Ultra II Directional RNA Library Prep Kit  according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system . Library sequencing was performed on an illumina Hiseq instrument with 150 bp paired end reads .Immediately after delivery, the placenta was thoroughly rinsed with sterile ice-cold phosphate buffered saline, cut into appropriately sized pieces (1.0 cm× 1.0 cm× 0.5 cm), and placed in RNAlater solution or 4% paraformaldehyde. The membranes near the breach were peeled from the placentas, and stored at -80°C for further experiments.RNA extraction and Real-time PCR (RT-PCR) were performed as described previously . TRIzol For IHC, after antigen retrieval using EDTA, the specimens were blocked with goat serum for 20 minutes before applying the primary antibody. Specimens were incubated with anti-NLRC4  for 12 hours in 4°C. Next, the sections were washed twice and subsequently incubated with HRP-polymer-conjugated secondary antibody  at room temperature. Finally, the specimens were then stained with 3,3-diaminobenzidine solution and haematoxylin. The slides were photographed with an inverted microscope .Immunofluorescence assay was performed as described in research by Hou et al . SectionCulture medium samples: The culture medium of RAW264.7 cells was collected, and the levels of NLRC4  in the culture medium were detected by enzyme-linked immunosorbent assay. Blood samples: Before labor, 10 ml of peripheral blood was collected in ethylenediaminetetramine Acetic acid (EDTA) vial. Blood samples were centrifuged (600g/min) for 10 minutes, serum was collected and stored at -80 environment for ELISA assay. The levels of NLRC4 , IL-1β , IL-6 , IL-18  and TNF-α  in the culture medium were detected by enzyme-linked immunosorbent assay2 or Fisher’s exact tests were used to compare categorical variables. Analyses were performed using SPSS software . P<0.05 was considered as significant difference.Numerical data are expressed as the mean ± SEM. Two independent sample data sets were tested using 2-tailed Student’s t test. Multiple group comparisons were evaluated by 1-way ANOVA followed by least significant difference t test for post hoc analysis. χHierarchical clustering shows differentially expressed mRNAs in membrane tissues of TPROM patients and the membrane tissues of non-TPROM pregnant (fold change (FC) < 0.5 or > 2, and P < 0.05) . ScatterP<0.001) and fetal membrane tissues of patients with TPROM (P<0.01) (P<0.05) (P>0.05) (P>0.05)   and AIM2(P<0.05)  inflamma(P>0.05)  and NOD2(P>0.05) . Immunoh(P>0.05)  membrane(P>0.05)  of patieP<0.001). Furthermore, compared to the control group, the mRNA abundance of caspase-1 was significantly higher in the fetal membranes of TPROM patients  (P>0.05)  . There w(P>0.05) . Immunof(P>0.05) .P<0.01), IL-18  and TNF-α  in the fetal membranes of patients with TPROM were significantly increased. While there was no significant difference in the expression of IL-6  in the fetal membranes between the two groups of pregnant women.Given that the pathogenesis of PROM is basically related to the inflammatory response and the inflammasome-mediated caspase-1 activation is involved in downstream secretion of the pro-inflammatory cytokines, such as IL-1β and IL-18, we investigated the levels of secreted proinflammatory cytokines in the different groups . The resP = 0.01) and caspase-1  gene expression with PROM.In P<0.01)  .P<0.05) and fetal membranes  of patients with TPROM combined with HCA was slightly higher than that of patients with TPROM with non-HCA. Overall, we conclude that high levels of NLRC4 may be one of the hallmarks of PROM.It is worth noting that 6 women in the PROM group were diagnosed with TPROM combined with Histological chorioamnionitis (HCA). Considering that HCA is the main cause and common complication of PROM, we further analyzed the relationship between HCA and NLRC4 content in serum of patients with TPROM. As shown in The main findings of this study are that NLRC4 plays an important role in the occurrence and development of TPROM, and that the level of NLRC4 in maternal serum is of great value in predicting the onset of TPROM.PROM not only causes chorioamnionitis, septicemia, placental abruption, and endometritis, but premature birth caused by PROM is also harmful to the newborn's health, such as causing neonatal ventricular bleeding, softening of white matter around the ventricle, and cerebral palsy , 20. TheAmniocentesis and amniotic fluid culture are recommended by the American Academy of Obstetrics and Gynecology for diagnosing amnion infection and indirectly for predicting PROM . HoweverThe mechanisms responsible for TPROM have not been elucidated, but are thought to involve inflammation. In addition to NLRC4, we also observed upregulation of caspase-1, ASC, IL-1β, and IL-18 in the membranes of patients with TPROM. In mechanism, the classic NLRC4 inflammasome pathway, that is, after binding to the ligands, the NLRC4 inflammasome is activated, resulting in its oligomerization to form multi-protein inflammasome complexes, which serves as a platform for recruitment, cleavage, and activation of caspase-1. Caspase-1 would then cleave pro-IL-1b, pro-IL-18, into their mature secreted forms of the cytokines –25. HereNLRs are usually activated by pathogen-associated molecules patterns. TPROM combined with HCA is not uncommon in obstetrics , 30. In In summary, NLRC4 is a markedly up-regulated gene in TPROM fetal membrane tissue, suggesting that NLRC4 is involved in the occurrence and development of TPROM. In addition, NLRC4 levels in maternal blood serum are closely related to TPROM and have the potential to assist doctors in predicting and diagnosing PROMS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Vibrio harveyi. Quorum sensing (QS) is a cell-to-cell communication system among bacteria that involves small signaling molecules called autoinducers, enabling bacteria to sense the surrounding environment. The autoinducers cause alterations in gene expression and induce bioluminescence, pigment production, motility and biofilm formation. The effect of CBG was tested on V. harveyi grown under planktonic and biofilm conditions. CBG reduced the QS-regulated bioluminescence and biofilm formation of V. harveyi at concentrations not affecting the planktonic bacterial growth. CBG also reduced the motility of V. harveyi in a dose-dependent manner. We further observed that CBG increased LuxO expression and activity, with a concomitant 80% downregulation of the LuxR gene. Exogenous addition of autoinducers could not overcome the QS-inhibitory effect of CBG, suggesting that CBG interferes with the transmission of the autoinducer signals. In conclusion, our study shows that CBG is a potential anti-biofilm agent via inhibition of the QS cascade.Cannabigerol (CBG) is a non-psychoactive cannabinoid naturally present in trace amounts in the Cannabis plant. So far, CBG has been shown to exert diverse activities in eukaryotes. However, much less is known about its effects on prokaryotes. In this study, we investigated the potential role of CBG as an anti-biofilm and anti-quorum sensing agent against  Bacteria can communicate by means of signaling molecules called autoinducers (AIs) in a process called quorum sensing (QS). Quorum sensing governs several processes which are critical for bacterial survival and allows them to respond to changes in cell density . These pVibrio harveyi produces at least three AIs, harveyi autoinducer 1 , autoinducer 2 , and cholera autoinducer 1 -3-aminoundec-2-en-4-one) that interact with their respective receptors LuxN, LuxP/Q and CqsS   . The qrr(qrr1-5) . Therefo(qrr1-5) .Cannabis sativa plant component cannabigerol (CBG)   using thmmalians . Specifimmalians , and hasmmalians . In addimmalians  and attemmalians  in mice. strains . Here weCannabigerol (CBG)  was purchased from NC Labs (Czech Republic) and dissolved in ethanol at a concentration of 18 mg/ml. As control, respective dilutions of ethanol corresponding to the different CBG concentrations were used. (S)-4,5-Dihydroxy-2,3-pentandione  was purchased from OMM Scientific Inc.V. harveyi wild-type strain BB120 was obtained from ATCC (Vibrio campbellii BAA-1116TM). The mutant bacterial strains were generously provided by Prof. B. Bassler (Princeton University)  medium  at 30°C for 20–24 h. For biofilm formation, the bacteria were grown in complete AB medium supplemented with higher concentrations of thiamine (0.3 mg/ml) and riboflavin (0.3 μg/ml).versity) . For plaV. harveyi 16S rRNA  as per the manufacturer’s instructions.DNA extraction and quantification were performed as previously described . Briefly16S rRNA  using a 595∼0.7) of wild-type V. harveyi (BB120) and mutant V. harveyi strains were diluted 1:200 in complete AB medium, and 180 μl of the diluted bacterial cultures were transferred to each well of an optic flat-bottom white 96-well cell culture microplate , together with 20 μl of CBG at different concentrations. The absorbance (OD595) and luminescence were read in parallel every 30 min for 20 h using an infinite M200PRO TECAN plate reader. The plate reader was kept at a constant temperature of 30°C throughout the experiment. The luminescence values, representing quorum sensing, were normalized by dividing them with the respective absorbance values in order to correct for differences in growth rates. The area under the curve was calculated for each sample and compared with untreated control samples as previously described (Overnight cultures (ODescribed .V. harveyi (OD595∼0.7) was diluted 1:10 in complete AB medium and incubated at 30°C under aerobic conditions in the absence or presence of 2 μg/ml CBG for 10 h. Eight milliliter of bacterial culture were used for each sample. At the end of incubation, the supernatant was removed, and the cells were washed with 2 ml of PBS and incubated with 2 ml of RNA Protect  for 5 min at room temperature. RNA was isolated using the RNeasy MINI kit (Qiagen) including on-column DNase digestion according to the manufacturer’s instructions. RNA purity and quantity were determined using Nanodrop . Only samples with an OD260/OD280 ratio of 2 and an OD260/OD230 ratio above 2 were used for cDNA synthesis. The samples were stored at −80°C until use.The assay was performed similarly to the method used by Ct−ΔΔ method. Gene expression was expressed in relative values, setting the expression level of the untreated control to 1 for each gene (The purified RNA was reverse transcribed into cDNA using the cDNA qScript cDNA synthesis kit (QuantaBio). The relative expression levels of target genes were analyzed by Bio-Rad CFX Connect Real-time system and the Bio-Rad CFX Maestro program. Power Sybr Green PCR Master mix (Applied Biosystems) was used to amplify the genes of 10 ng cDNA per well in combination with 300 nM of respective F/R primer set . For eacach gene .V. harveyi mutant strains in incomplete AB medium  was collected, filtrated (0.22 μm) and kept at –20°C. The BB152  was used for obtaining AI-2, while MM30  was used for preparation of AI-1. Supernatant of MM77  was used as a negative control.Supernatant from log-phase growing V. harveyi culture (OD595∼0.5) were inoculated at the center of the agar plate and incubated at 30°C for 15h. The area of the motility halo was measured using Image J software  and compared with the control.The motility assay was performed on soft agar plates as described previously . BrieflyBiofilms were prepared as described above with different concentrations of CBG and corresponding ethanol dilutions. Biofilms without CBG served as additional controls. Biofilm was allowed to form on sterile circular glass pieces that were inserted into the wells of 96-well microtiter plates. After a 24 h incubation, the glass specimens were removed, rinsed with DDW and fixed in 4% glutaraldehyde for 40 min. The glass specimens were washed again with DDW and allowed to dry at room temperature. The specimens were then mounted on a metal stub and sputter coated with gold prior to SEM analysis. A high-resolution scanning electron microscope  was used. Three specimens from each treatment group were prepared and examined under SEM to evaluate the effect of CBG on biofilm formation .t-test in Microsoft Excel. A p < 0.05 was considered significant.Three independent experiments were conducted and triplicate samples were measured within each experiment. Statistical analysis of the collected data was performed using Student’s V. harveyi (wild-type strain BB120), the bacteria were exposed to different concentrations of CBG or the respective ethanol dilutions in complete AB growth medium and incubated for 20 h. Untreated V. harveyi served as an additional control. The planktonic growth was monitored by measuring the optical density (p < 0.05). Strong inhibition was already observed at 1 μg/ml CBG, with only a slightly higher inhibition when increasing the concentration to 20 μg/ml . To further confirm the anti-biofilm activity, biofilms of V. harveyi formed in the absence or presence of 20 μg/ml CBG or respective ethanol dilutions were examined under a scanning electron microscope (SEM) at a magnification of x1000–5000. As can be seen from Bacterial biofilm formation is associated with the quorum sensing process which can be critical for bacterial survival . To testV. harveyi was studied. The motility halos decreased with increasing concentrations of CBG in a dose-dependent manner (p < 0.05). Ethanol as high as 2% had no effect on the motility  strain  strain  strain  and the ) strain . DPD is ) strain .V. harveyi was incubated in the absence or presence of 2 μg/ml CBG for 10 h, and the RNA was extracted for real-time PCR analysis. We observed that both the luxU and luxO gene expression were strongly induced (x3.5–4.5-fold induction)  . The simultaneous upregulation of AphA further antagonizes LuxR gene and protein expression  and receptors  are shown in luxQ gene was highly induced, while luxM, luxN, and luxP were modestly induced. CBG had barely any effect on the luxS gene expression  . Also, tduction) , indicatpression . The exppression .V. harveyi deficient in autoinducer production and/or autoinducer receptors. The bioluminescence vs. planktonic growth was studied for each strain in the absence or presence of 1 or 2 μg/ml CBG . These findings suggest that CBG predominantly antagonizes AI-2, which is the major autoinducer during the exponential growth phase of V. harveyi . In contrast to the AI-2 receptor null mutants, the mutant lacking luxN, the receptor for AI-1, and the mutant lacking both luxN and luxQ, showed diminished response to CBG (p < 0.05), suggesting that LuxN is a major receptor targeted by CBG.In a further search for understanding the anti-quorum sensing activity of CBG, we used different mutants of g/ml CBG . The luxild-type , whereas harveyi . Interesild-type , suggest harveyi , and no luxM null lacking AI-1 showed a 40% reduction in bioluminescence in comparison to wild-type, while the luxS null lacking AI-2, showed more than 98% reduction in bioluminescence  than the wild-type   . Medicinranberry .V. harveyi. We demonstrated that CBG shows strong anti-quorum sensing and anti-biofilm activities on V. harveyi at concentrations that do not affect their viability. In addition, CBG caused strong reduction in their motility. So far, the activities of CBG have mainly been studied in eukaryotes where it has been found to be neuroprotective, acting as an anti-oxidant  mutant showed 98% less basal bioluminescence in comparison to wild-type, although it still emits bioluminescence. The luxM null (AI-1–) mutant showed a 40% reduction in basal bioluminescence in comparison to wild-type. Interestingly, CBG did not reduce the bioluminescence of the luxS null mutant, while still had a significant effect on the luxM null mutant. This means that the major anti-quorum sensing effect of CBG acts on interfering with the AI-2-dependent signals.Quorum sensing has been shown to enhance the flagellar-dependent motility of  harveyi , and the harveyi . Therefor stages . The luxluxP and luxQ null mutants deficient in the AI-2 receptor still responded well to the anti-QS activity of CBG. These mutants differed from the luxS mutant in still showing high bioluminescence, which might be due to the lack of agonism of the AI-2 receptor on LuxU/LuxO. The luxP and luxQ mutants rely mainly on the AI-1/LuxN for the induction of bioluminescence suggesting that it is this pathway that is targeted by CBG. We observed that the luxN null mutant responded less well to CBG than wild-type, providing further support that LuxN is a target of CBG. It is worth noting that the LuxN has nine transmembrane regions (luxU and luxO as well as the LuxO-regulated genes aphA, hfq and the small regulatory RNAs qrr1-5, demonstrating that CBG does increase the LuxO activity in V. harveyi. Concomitant with the increased LuxO activity, the LuxR gene expression was strongly reduced by CBG, which explains the anti-quorum and anti-biofilm activity of CBG (A surprising result was the observation that the  regions  and CBG  regions . It couly of CBG .Cannabis component CBG on QS-mediated processes in V. harveyi, including bioluminescence, biofilm formation and motility, at concentrations that do not affect planktonic growth. CBG antagonizes both the AI-1 and AI-2 pathways, through acting, among others, on LuxN with resulting increase in LuxO activity. The interference of CBG with the bacterial signal-transduction system provides a novel innovative way to combat bacterial biofilm formation.In summary, the present study demonstrates the antagonistic effects of the The datasets generated for this study are available on request to the corresponding author.MA, RG, RS, BZ, MF, and DS conceived the idea. MA designed and performed the experiments and analyzed the data. MA wrote the paper with RS and DS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the effect of an exercise prescription and a 1-year supervised exercise intervention, and the modifying effect of the family history of type 2 diabetes (FH), on long-term cardiometabolic health.For this prospective randomized trial, we recruited non-diabetic participants with poor fitness . Participants were randomly assigned with stratification for FH either in the exercise prescription group  or the supervised exercise group  group and compared with a matched control group from the same population study . The PG and EG received exercise prescriptions. In addition, the EG attended supervised exercise sessions two times a week for 60 min for 12 months. Cardiometabolic risk factors were measured at baseline, 1 year, 5 years, and 6 years. The CON group received no intervention and was measured at baseline and 6 years.The EG reduced their body weight, waist circumference, diastolic blood pressure, and low-density lipoprotein-cholesterol (LDL-C) but not physical fitness (p=0.074) or insulin or glucose regulation (p>0.1) compared with the PG at 1 year and 5 years (p≤0.011). The observed differences were attenuated at 6 years; however, participants in the both intervention groups significantly improved their blood pressure, high-density lipoprotein-cholesterol, and insulin sensitivity compared with the population controls (p≤0.003). FH modified LDL-C and waist circumference responses to exercise at 1 year and 5 years.Low-cost physical activity programs have long-term beneficial effects on cardiometabolic health regardless of the FH of diabetes. Given the feasibility and low cost of these programs, they should be advocated to promote cardiometabolic health.NCT02131701.ClinicalTrials.gov identifier  Family history of type 2 diabetes is associated with reduced physical fitness and a lower expression of genes in the oxidative phosphorylation.Limited prospective data are available on how type 2 diabetes or family history of type 2 diabetes affects physical fitness or metabolic response to exercise.Family history of type 2 diabetes attenuated the effect of exercise on waist circumference and low-density lipoprotein.One-year low-cost structured exercise intervention and exercise prescription intervention similarly led to an improved cardiometabolic health 6 years later compared with the population-based control group.Individuals with family history of type 2 diabetes can in most parts expect similar cardiometabolic health benefits from regular exercise; however, the diminished responsiveness to waist circumference and low-density lipoprotein warrants further investigation.Physical activity prescription could be a feasible and readily available option to promote long-term cardiometabolic health in low fit populations.2max), insulin sensitivity, adiposity, LDL-cholesterol (LDL-C), and blood pressure.6–8Regular physical activity has been associated with a myriad of health benefits including reduced risk of mortality, diabetes and other non-communicable diseases.2max training response is strongly explained by heritability (47%), but only minimally by age, sex, and ethnic origin.16 17Although physical activity has a central role in the prevention and treatment of type 2 diabetes, the interindividual variability in response to exercise is large.Therefore, this study aimed to investigate the long-term (5 years) effect of exercise prescription versus a 1-year supervised exercise intervention (EG) on aerobic physical fitness, muscle strength, and cardiometabolic parameters in subjects with poor physical fitness, stratified for FH. We also compared the change in measured outcomes between the intervention groups and matched control subjects originating from the same population study at 6 years.The Prevalence, Prediction and Prevention of diabetes (PPP)-Botnia Study is a population-based study (n=5 208) in Western Finland initiated to obtain accurate estimates of prevalence and risk factors for diabetes, pre-diabetes and the metabolic syndrome in the adult population and to use this information for prediction and prevention of the disease.In addition to the exercise intervention groups, a population-based control group  was recruited from the participants in the PPP-Botnia Study. The CON group received no specific intervention. Male and female participants were considered eligible for the CON group if they were (1) non-diabetic and aged 30–70 years, (2) had poor physical fitness based on a 2 km walking test  and (3) participated in both the baseline and 6-year follow-up study. The CON group was subdivided according to the FH by same criteria as the intervention groups. The baseline visits occurred 2004–2008 and 6-year follow-up visits 2010–2015. The 10.1136/bmjdrc-2020-001377.supp1Supplementary dataAll study subjects participated in two individual sessions of exercise prescription aiming at 30 min of moderate exercise at least 5 days a week. They also received exercise diaries. Participants in the PG performed unsupervised training, while participants in the EG were offered supervised physical training in groups twice a week during 12 months. The supervised training included both endurance training  and resistance training in a gym. The training sessions lasted 60 min including warm-up. To control the intensity of aerobic training, 70%–85% of age-adjusted maximal pulse rate was recommended, and heart rate monitoring was used periodically. Muscle strength training was performed in two sets of 12–15 repetitions focusing on major muscle groups. The first set (warm-up) was performed with 10 repetitions at 50% of repetition maximum, and the second set at 70%. Resistance was progressively increased according to the repeated testing of muscle strength. Experienced trainer supervised the training and also performed the testing of the muscle strength.In the PG and EG, all measurements were taken at baseline, 1 year, and 5 years. All measurements were also collected from the CON group at baseline. At 6-year follow-up, body composition, waist circumference, blood pressure, blood lipid and glucose metabolism were measured from all groups .Aerobic physical fitness as a primary outcome was assessed in all subjects by a 2 km walking test (UKK), which provides an indirect estimate of oxygen uptake.Muscle strength  was assessed by fitness equipment  after a 5–10 min warm-up. Each subject performed five repetitions during 10 s. The resistance was increased until the subjects were unable to perform more. One repetition maximum (1 RM) was calculated based on the Brzyckis formula: 1 RM = weight/(1.0278 – (0.0278 × repetitions)).Body weight  and height were measured with subjects in light clothing without shoes, and BMI was calculated. Waist circumference was measured with a soft tape on standing subjects midway between the lowest rib and the iliac crest. Two blood pressure recordings (Omron 711) were obtained from the right arm of a sitting person after 30 min of rest at 5 min intervals, and their mean value was calculated.Questionnaires were used to obtain information about other diseases, current medication, smoking and alcohol consumption. At baseline, 1 year and 5 year follow-up frequency and intensity of physical activity during the past 12 months was assessed using the validated Kuopio Ischemic Heart Disease questionnaire (KIHD).The subjects participated in an oral glucose tolerance test (OGTT) by ingesting 75 g of glucose  after a 12-hour overnight fast. Samples for the measurement of plasma glucose and serum insulin were drawn at 0, 30, and 120 min. Glucose and insulin area under curve  were also calculated. Fasting samples were drawn for the measurement of serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), and triglyceride concentrations. LDL-C concentrations were calculated using the Friedewald formula.25Serum insulin concentrations were measured with a fluorometric immunoassay , plasma glucose by HemoCue Glucose 201-assay , and serum lipids by an immunoturbidometric method . Insulin sensitivity was assessed with homeostasis model assessment for insulin resistance (HOMA-IR=[(fasting serum insulin concentration/fasting plasma glucose concentration)/22.5]According to power analysis, 71 participants in each group were required to detect (power 80% and 95% CI) a 20% difference between the groups in achieving a 15% improvement in physical fitness. All data are reported as mean (SD or 95% CIs).General linear models were used to compare the baseline characteristics between the groups (EG and PG stratified for FH). Linear mixed models were applied to test the differences between the PG and the EG during the 1-year and 5-year follow-up. The independent variables for these models were group (EG vs PG), time , and FH and their interaction (group × time × FH). This model was employed to test the impact of FH on exercise responses (group × time × FH interaction effect) and the differences between the EG and PG (time × group interaction effect). Subjects who had data on all time-points for a specific variable were included in the analyses. Nominal p values are reported, and alpha level 0.05 was set as a threshold for statistical significance. Pearson correlation was used to measure correlation between the exercise dose during the 1-year intervention and the residual change in outcomes from baseline to 1 year.The marginal mean weighting through stratification (MMWS) method was applied to estimate the average treatment effect between the intervention groups (PG and EG) and the CON group.The 6-year response was compared between groups with and without FH and adjusted for baseline values. To correct for multiple comparisons , a p value <0.0125 (=0.05/4=0.0125) was used as a threshold for statistical significance in these analyses.The generalized estimating equations were applied to compare the change in usage of medication for blood pressure and cholesterol between the CON, PG, and EG from baseline to 6-year follow-up. Penalized maximum likelihood logistic regression (Firth’s logistic regression) was used to compare the diabetes medication between the groups at 6 years, as none of the participants had diabetes medication at baseline, and the frequencies were low at 6 years.All analyses were performed with Stata/SE V.14.2, Stata/MP V.15.1, and Stata/MP V.16.1.A total of 290 participants were randomized into the two intervention arms. Altogether 266 participants (136 in PG and 130 in EG), who completed the 5-year follow-up, were included in the primary analyses . BaselinDuring the 1-year intervention, 44% of the participants in the EG trained on average at least once and 11% trained at least twice per week. No significant differences in the dose of the intervention was observed between the FH− and FH+ groups (p>0.1) . AccordiThe EG had significantly lower body weight and waist circumference at 1 year, as well as lower total cholesterol, LDL-C, and diastolic BP  and bettAt 6 years , there were no significant differences between the PG and EG in change any of the outcomes (p>0.079) , which iThe baseline characteristics were similar between the CON and the pooled intervention group, except for 0.1 mm Hg (95% CI 0.04 to 0.2) lower fasting glucose, 2.6 mmol/L (95% CI 1.3 to 4.0) lower diastolic blood pressure, and 8.1 IU/L (95% CI 2.3 to 13.8) higher 30 min insulin level in the pooled intervention compared with the CON group .Compared with CON group, the pooled intervention group had significantly lower systolic and diastolic blood pressure, insulin levels, HOMA-IR, and higher HDL-C at 6 years  and a noFH modified the response of lipids and waist circumference to training. In FH− individuals, EG improved LDL-C at both 1 and 5 years and waist circumference at 1 year compared with the PG . No suchAt 6 years, there was a significant interaction between the mode of intervention (EG and PG) and FH on the change in LDL-C (group × FH interaction effect) . Among tIn an adult non-diabetic population with low cardiorespiratory fitness, we detected no difference in cardiorespiratory fitness between the 1 year EG or PG interventions at 1 or 5 years. The 1-year EG intervention, however, induced greater improvement in muscle strength, larger reduction in body weight and diastolic blood pressure, and LDL-cholesterol than the prescription only intervention. In addition, contrary to our hypothesis, we found no strong evidence that FH modifies individual responsiveness to exercise. Our findings suggest that individuals with FH may be less prone to reduce their LDL-C and possibly waist circumference with exercise, but overall, they achieve similar exercise-induced improvements in insulin resistance, HDL-C, and blood pressure as individuals without FH. The main finding was that irrespective of the mode of intervention, the 1-year low-cost exercise interventions resulted in a lower 6-year systolic and diastolic blood pressure, insulin levels, HOMA-IR and higher HDL-C compared with the background population, the CON group, taken from the same population study. Thus, the present findings support the conclusion that both EG and PG interventions have beneficial long-term effects on cardiometabolic health independent of the FH status.The strength of the study is the population-based recruitment of the study participants and random allocation of participants into a training and prescription group. We also observed a very low overall dropout rate, as 91.7% of the participants participated in the 5-year examination. We were able to take a real-life control group from the PPP-Botnia cohort and to estimate the propensity score based average treatment effect of those who did or did not receive the intervention. Furthermore, participants reported no major adverse effects that can be traced back to the interventions. Thus, these interventions can be safely recommended for adult populations.There are some limitations of the current study that should be addressed. As in many other studies, we encountered problems with monitoring physical activity and quantity and intensity of exercise interventions. The key instruments were 12-month recall questionnaire, the exercise diary and adherence to weekly exercise sessions, but this was left to the individual’s compliance. Although we aimed to test a less resource-consuming intervention, in future studies, it may be beneficial to include activating contacts, including new web-based technologies, by health professionals or trainers to increase the motivation and adherence to the intervention.In the present study, individually given low-resource intensive exercise prescription intervention aiming for 150 min of exercise per week provided similar long-term cardiometabolic health benefits as the supervised exercise intervention in low-risk population. Participants that received either type of exercise intervention (PG+EG) improved their blood pressure, HDL-C, and insulin sensitivity, without a significant change in the waist circumference or body weight, compared with the CON group during the 6.8-year follow-up . Our finBoth the PG and the EG performed similarly in most of the measured variables. The EG gained more muscle strength during the intervention, which can be explained by the fact that resistance training was the most common type of exercise . Also, m2max between the first-degree relatives of type 2 diabetic patients and controls after a 10-week aerobic exercise intervention.2max gain as the FH− group.2max is infeasible. In fact, the walk-test based predicted fitness score correlates well with direct VO2 measurements, and it can be successfully applied in follow-up studies.15FH did not influence aerobic fitness response to training. These findings are consistent with a previous study reporting similar increase in the VO2max,Previous studies have well established that regular exercise decreases waist circumference and reduces LDL-C level.In conclusion, 1-year PG or EG intervention provide similar long-term cardiometabolic health benefits compared with the community-based control population. Although the FH modulated the LDL-C and waist circumference response to exercise, it seems that it does not systematically modify cardiometabolic response to exercise in any major way. These findings illustrate that low-resource promotion of physical activity in a clinical setting can be a feasible tool for enhancing long-term cardiometabolic health in an adult FH− or FH+ populations. We encourage healthcare policymakers and practitioners to take actions that promote incorporation of low-cost physical activity programs into the clinical practice.